Dao Lienas Traansgenicas De Guisantes

MPMI Vol. 13, No. 9, 2000, pp. 922–928. Publication no. M-2000-0620-01R. © 2000 The American Phytopathological Society

Construction of Transgenic Pea Lines with Modified Expression of Diamine Oxidase and Modified Nodulation Responses with Exogenous Putrescine
J.-P. Wisniewski and N. J. Brewin John Innes Centre, Norwich, NR4 7UH, U.K. Accepted 12 May 2000.
Diamine oxidase (DAO) mightinfluence pea nodule development either by regulating the peroxide-driven crosslinking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity inextracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing cosuppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in thecrosslinking of matrix glycoprotein, indicating that putrescinederived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serveto reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues. Additional keywords: extensin, legume, Rhizobium leguminosarum bv. viciae, symbiosis, transformation. Diamine oxidases (DAO) are copper-containing enzymeswidely distributed in microbial, plant, and animal systems. DAO catalyzes the oxidative deamination of diamines such as putrescine with the formation of the corresponding aldehyde along with hydrogen peroxide and ammonia. Some plant DAOs have been purified and characterized, especially from leguminous species, in which DAO occurs at high concentraCorresponding author: N. J. Brewin; Telephone: +4401603 450273; Fax: +44 01603 450045; E-mail: brewin@bbsrc.ac.uk Current address of J.-P. Wisniewski: Aventis CropScience, BP 9163, 69263, Lyon 09, France.

tion. The enzymes are loosely associated with plant cell walls, and high activity has been reported in the middle lamellar region and in the vascular parenchyma cells of pea seedling roots, as well as in lignified tissues (Medda et al. 1995;Wisniewski et al. 2000). In situ hybridization studies indicate that DAO transcript is abundant in all infected tissue of pea nodules, both in the invasion zone and in the host cells that support nitrogen-fixing bacteroids (Wisniewski et al. 2000). The function of DAO may be due in part to the modulation of the level of polyamines (PAs) and in part to H2O2 production through the oxidation ofdiamines. In chick-pea, the level of diamines is apparently controlled by catabolism rather than synthesis (Torrigiani and Scoccianti 1995), i.e., through oxidation by DAO. Hydrogen peroxide, the product of this reaction, can be utilized by peroxidase enzymes, thus leading to the modulation of cell wall plasticity. Spatial and temporal correlations of DAO and peroxidase activity have suggested a rolefor DAO in cross-linking of proteins and polymerization of monolignols (Angelini et al. 1990; Moller and McPherson 1998). Cross-linking of proteins like extensins occurs not only during normal plant growth but also in response to stress and wounding. The activity of DAO is modulated by wounding and other stress factors (Maccarrone et al. 1997; Smith and Barker 1998). Recently, it has been shown...